Ovicidal and repellent activities of several plant essential oils against Periplaneta americana L. and enhanced activities from their combined formulation

Natural ovicidal and repellent agents against Periplaneta americana L. are urgently needed, and plant essential oils (EOs) can assume this role quite readily. In this study, ovicidal and repellent activities against Periplaneta americana of EOs from Cymbopogon citratus (Stapf.), Cinnamomum verum (J. Presl.), Eucalyptus globulus (Labill.), Illicium verum (Hook.f.), and Zanthoxylum limonella (Alston) in soybean oil and in ethyl alcohol were determined by topical and dual-choice assays, as well as 10% cypermethrin and a combined formulation of 5% C. verum EO + 5% I. verum EO. Cypermethrin at 10% provided the highest toxicity (100% inhibition rate) against the eggs, but only slightly higher than that (99.3%) provided by the combined EO formulation, while the highest repellent activity against the adults was provided by the combined formulation (89.5% repelled cockroaches at 48 h after treatment). In addition, all EO formulations in soybean oil provided higher ovicidal and repellent activities than those in ethyl alcohol. To conclude, the combined EO formulation in soybean oil can replace cypermethrin because their efficacy was nearly equivalent, but the combination should be much safer to use.

The inhibition rates of every treatment and control against P. americana eggs at 30 days after exposure are tabulated in Table 2. All treatments and controls were 10% solutions of a compound in soybean oil and in ethyl alcohol. The inhibition rate of every EO formulation in soybean oil (85.3-96.7%) was higher than that of the same EO in ethyl alcohol (60.3-90.1%). The highest egg inhibition rate, at 96.9%, among all 10% EO solutions in soybean oil, was exhibited by I. verum EO, and the lowest egg inhibition rate, at 85.3%, was shown by C. verum EO. The highest inhibition rate among all 10% EO formulations in ethyl alcohol, at 90.1%, was achieved by Z. limonella EO, and the lowest, at 60.3%, was exhibited by C. citratus EO. The implications of these highest and lowest inhibition rate results are separately discussed in the discussion section. In contrast, 10% w/v cypermethrin both in soybean oil and in ethyl alcohol exhibited a full 100% inhibition rate, the highest among all treatments. Regarding the inhibition rate index (IRI), every EO formulation showed an IRI of less than 1 (0.6-0.97), i.e., they were less toxic against P. americana eggs than 10% w/v cypermethrin.
The percentage repellent rates against P. americana adults at 48 h of exposure of treatments and control in soybean oil and in ethyl alcohol are shown in Table 3. All 10% solutions in soybean oil of the five plant EOs exhibited a high percentage range of repellent activity, of 60.8-100%, against P. americana adults, and each of them exhibited a higher percentage repellent activity than the same EO in ethyl alcohol (47.6-88.2%).
There was an inverse relationship between percentage repellent activity and time of exposure. As the exposure time increased, the repellent activity percentage decreased, as shown by the regression curve in Fig. 3 Table 4. As indicated by the ERI, all EO formulations were more repellent against P. americana adults than 10% cypermethrin. The ERIs of all EO formulations were always higher than that of cypermethrin at any time of exposure. The highest ERI observed was 1.71, achieved by 10% C. verum EO in soybean oil after 48 h of exposure, signifying that this EO formulation was 1.7 times more potent than 10% cypermethrin. Other EO formulations showed an ERI in the range of 1.09-1.62.
As shown in Tables 2 and 5 and, the ovicidal inhibition rate exhibited by the combined formulation was considerably higher (99.3%) than that exhibited by 10% I. verum EO (91.2%). In the same vein, the repellent Table 1. Chemical constituents of Cymbopogon citratus, Cinnamomum verum, Eucalyptus globulus, Illicium verum, and Zanthoxylum limonella and their percentage yield in the total chemical profile. a R = Retention index of a chemical constituent determined with an HP-5 MS column and compared with the retention indices of standard alkanes (C 7 -C 30 ) for identity verification. b K = Kovat retention index from NIST17 62 . c I = Identified and confirmed by mass spectrum (M) matching with chemicals in the computer mass library of Adams 61 and by retention index (R) matching with those reported in NIST17 62 .

Item
Compound Peak area (%), (average of 3 runs ± SD)  Table 6) was higher (89.5%) than that exhibited by C. verum EO (83.4%) ( Table 3). Moreover, in both ethyl alcohol and soybean oil solvents, the formulation of the 5% I. verum EO + 5% C. verum EO combination exhibited a range of inhibition rates, 91.9-99.3%, which was very close to the 100% inhibition rate of cypermethrin. The most significant result was that the IRI of the combined EO formulation in soybean oil was very close to one (i.e., 0.99), indicating that its potency was almost identical to that of cypermethrin. Finally, the combined formulation in soybean oil (99.3% ovicidal inhibition rate) exhibited a higher efficacy than the combined formulation in ethyl alcohol (91.9% inhibition rate). As shown in Tables 3 and 6 and, the percentage repellent activity exhibited by the combined formulation was considerably higher than that exhibited by 10% C. verum EO and 10% cypermethrin. The combined formulation of 5% I. verum EO + 5% C. verum EO exhibited the highest repellent activity of 89.5% at 48 h of exposure, while 10% cypermethrin showed 44.7% repellent activity. The most significant result of this study was that the ERI of this combined EO formulation in both soybean oil and ethyl alcohol solvent was in the range of 1.47-1.94, signifying that this combined formulation was 1.47-1.94 times more potent in repellent activity than 10% cypermethrin. Regarding the influence of solvent on efficacy, following the same trend of influence on ovicidal inhibition  www.nature.com/scientificreports/ rate mentioned above, the repellent activity exhibited by the combined formulation in soybean oil (89.5%) was higher than the activity exhibited by the combined formulation in ethyl alcohol (65.7%).

Discussion
Several factors affected the extraction yield of plant essential oil, such as agricultural practice, cultivation area, and extraction method 26 . The observed essential oil yields from the five plants in this study were checked and confirmed against the values reported in the literature, and mostly, they agreed very well. Namely, the essential oil yield of C. citratus observed in this study was 1.15% v/w, while papers by Soonwera and Sittichok 24 , Aungtikun et al. 25 , and Verma et al. 26 29 . The observed I. verum EO yield was 3.23% v/w, slightly lower than the 4.0-4.5% v/w, (water distillation) reported by Gholivand et al. 33 and the 4.0% v/w (water distillation) reported by Aungtikun et al. 25 , and the observed yield of Z. limonella EO was 9.27% v/w, slightly lower than the 9.63% v/w reported by Charoensup et al. 30 . Moreover, although local species of C. citratus in Thailand and in India have the same number of constituents, the percentages of the constituents in their chemical profile are different 34 . Therefore, those yield percentages were determined and checked against the values in recent literature. Many factors influence the percentage of a major constituent in an EO's chemical profile, such as plant maturity, harvesting time, and good harvesting practice 25 . The observed GC-MS chemical profiles of every EO were well-matched with those reported in a previous study. Namely, the percentage of geranial, the major chemical constituent of C. citratus EO observed in this study, was 46.33%, while papers by Soonwera and Sittichok 24 , Aungtikun et al. 25 , and Chauhan et al. 34 39 .
Nevertheless, there were some slight discrepancies, such as the yield and percentage in the chemical profile of the major constituents of I. verum and Z. limonella EOs. These discrepancies were likely due to the usage of different plant parts for EO extraction, a difference in the extraction procedures, and variations in genotypic, phenotypic, and agroecological factors (e.g., plant age, growth phase in plant development, amount of sunlight and moisture, as well as the temperature at the farm's geographical location 40 . High toxicity of plant EO in soybean oil against insect pests has been reported in recent literature. A study on EO toxicity against head louse eggs by Soonwera et al. 41 concluded that Zingiberaceae plant EOs in soybean oil exhibited high toxicity to the eggs of head louse (Pediculus humanus capitis; Pediculidae: Phthiraptera), with a full 100% inhibition rate. In this study, 10% I. verum EO and the combined EO formulation in soybean oil showed high egg inhibition rates of 96.9% and 99.3%, respectively, against P. americana. This result was expected before the experiment started because the outcomes of many studies on I. verum EO against eggs of insect pests other than P. americana have been reported in the literature. Specifically, Sinthusiri and Soonwera 42 reported that 10% I. verum EO exhibited markedly high oviposition deterrent and ovicidal activities against female houseflies (Musca domestica; Muscidae: Diptera), while Matos et al. 43 reported that I. verum EO exhibited a high inhibition rate against the eggs of cowpea weevil, Callosobruchus maculatus (Bruchidae: Coleoptera). In addition to a high repellent activity against the adults, 10% C. verum EO and the combined formulation in soybean oil also exhibited a high repellent activity against P. americana adults-100% and 83.4% and 100% and 89.5% repellent activity at 1 and 48 h after exposure, respectively. Many papers have reported a high repellent activity of C. verum EO against several insect pests such as Plodia interpunctella (Pyralidae: Lepidoptera), Bemisia tabaci (Aleyrodidae: Homoptera), Ae. aegypti (Culicidae: Diptera), Sitophilus zeamais (Curculionidae: Coleoptera) 44 , Sitophilus oryzae (Curculionidae: Coleoptera) 45 , and ant 46 .
Unlike a large number of papers on individual EOs against P. americana mentioned above, papers on combined EO formulations against P. americana are limited. An example is a paper by Zibaee and Khorram 16 reporting that a combined formulation of E. globulus + R. officinalis EOs exhibited a stronger insecticidal effect against adult P. americana than that of each individual EO component. Aungtikun et al. 25 reported that a combined formulation of 0.5% I. verum EO + 0.5% geranial showed a higher insecticidal effect against adult house flies (M. domestica) than each individual EO.
Most notably, all EO formulations in soybean oil exhibited higher ovicidal and repellent activities against P. americana eggs and adults than all EO formulations in ethyl alcohol, in full agreement with findings from Sittichok and Soonwera 47 that eight herbal EOs in soybean oil showed a higher inhibition rate against P. americana eggs than the same EOs in ethyl alcohol. Findings on repellent activity against adult mosquitoes in studies by Soonwera and Phasomkusosil 48 and Phasomkusosil and Soonwera 49 showed the same trend: 10% EOs from C. citratus, C. nardus, Cananda odorata, Ocimum basilicum, and Z. cassumunar in soybean oil weremore repellent and feeding deterrent against adult females of Anopheles minimus, Ae. aegypti, and Culex quinquefasciatus than the same EOs in ethyl alcohol. In the same vein, in this study, C. verum EO and the combined EO formulation exerted their potency better in soybean oil than in ethyl alcohol.
The soybean oil solvent in this study was pure soybean oil, but the ethyl alcohol solvent was 70% (v/v) ethyl alcohol in water. Therefore, the reason that a soybean oil solution of an EO is more repellent than the same EO in ethyl alcohol solution is that soybean oil is more lipophilic than ethyl alcohol, and a lipophilic solvent retards evaporation of EOs 49 . In this study, C. verum EO and the combined EO formulation in soybean oil provided a longer, more persistent repellent activity against P. americana than the same EO or combined EOs in ethyl alcohol, and the higher activity was attributed to this reason. On the other hand, an EO in a hydrophilic solvent evaporated faster and hence shorter, less persistent repellent activity. This claim is supported by a paper by Phasomkusosil and Soonwera 49 : 10% Citrus sinensis EO in soybean oil was more repellent (30-60 min) against adult females of Cx. quinquefasciatus, Ae. aegypti, and An. minimus than the EO in ethyl alcohol (less than 1.0 min). Another paper by Phasomkusosil and Soonwera 49 and Phukerd and Soowera 50 reported that 10% Z. cassumunar EO in soybean oil was more repellent (70 min) against adult females of Ae. aegypti than the EO in ethyl alcohol (30.0 min).
On the other hand, even though the reason that I. verum EO and the combined EO formulation were more potent in ovicidal activity in soybean oil than in ethyl alcohol is also the higher lipophilicity of soybean oil, but www.nature.com/scientificreports/ the mechanisms of action were not the same. The mechanism responsible for the higher ovicidal activity is that a lipophilic solvent can carry an EO through insect cuticles more readily because insect cuticles are mainly lipophilic 41 , hence more EO passes into the insect, providing more ovicidal activity from a higher amount of EO in its body 41,51,52 . This claim is supported by a conclusion from Rajashekar and Shivanandappa 53 and Jankowska et al. 54 that embryo and egg mortality were strongly affected by EO's high cuticle permeability. The outcomes of this experiment confirm the expectation that every EO in soybean oil was more effective than the same EO in ethyl alcohol. Regarding the high ovicidal activity (96.9-99.3%) against P. americana eggs of 10% I. verum EO among all tested individual EO formulations, this high activity is likely to be provided by its main constituent, transanethole 25 . Strong potency of trans-anethole against mosquitoes and houseflies was reported by Aungtikun et al. 25 and Pavela 55 . Its mechanism of action was reported in Jankowska et al. 54 and Bosch-Serra et al. 56 : namely, trans-anethole inhibited the development of embryo by blocking cytochrome P450 detoxification enzymes, retarding cell growth and juvenile hormone production and weakening the immune system, leading to eventual embryo death.
In the same way, the high repellent activity (100% at 1 h) against P. americana adults of 10% C. verum EO is likely to be provided by its main constituent, cinnamaldehyde 47 . Cinnamaldehyde has been reported to strongly repel P. americana adults (75.23%) 47 . The repellent activity of cinnamaldehyde is strong because it causes serious damage to their respiratory system 47 , so they always try to avoid it. This conclusion is supported by a claim by Devi and Devi 57 that the mechanisms of action of cinnamaldehyde against insect pests were inhibition of its respiratory system by enzyme inhibition, cell membrane alteration, and reduced cell membrane integrity, as well as reduced cell respiration. Because of these reasons, C. verum EO showing the lowest ovicidal efficacy (in Table 2) makes sense because it does not have a constituent that strongly inhibited the eggs of P. americana like trans-anethole that I. verum EO has, hence it provided a low inhibition rate against the eggs of P. americana and house fly, as reported by previous works of Sittichok and Soonwera 47 and Sinthusiri and Soonwera 42 . In the same vein, the graph of repellent activity of C. citratrus EO in Fig. 3 looks similar to that of C. verum EO but not that of I. verum EO because of them have geranial and cinnamaldehyde as their major constituent, while I. verum EO do not.
The overall findings from this study indicate that the combined formulation of 5% C. verum EO + 5% I. verum EO would be a good current alternative to cypermethrin because, first, it is equally or more potent than cypermethrin in ovicidal and repellent activities and, second, they should be less harmful to humans and non-target organisms and not be as persistent as cypermethrin in the environment. Findings supporting the first reason were as follows. First, against P. americana adults, C. verum EO and the combined EO formulation were 1.71-1.97 times more potent than cypermethrin in repellent activity, while the ovicidal activity of cypermethrin was only slightly more potent than those of I. verum EO and the combined EO formulation. Moreover, these findings are supported well by findings from a previous study by Sinthusiri and Soonwera 42 that 10% cypermethrin showed a 100% inhibition rate against house fly eggs, while 10% I. verum EO showed a 97.33% rate. Second, findings by Ichikawa 58 and Sharma et al. 59 support the safety reason for replacing cypermethrin with a plant essential oil: cypermethrin is seriously toxic to the nervous and immune systems of humans, especially pregnant women and children, while findings by Aungtikun and Soonwera 27 and Patra et al. 60 support the claim that EOs are quickly degraded in the environment. Moreover, its safety to humans has long been established in Southeast Asia because it has been widely used as folk medicine since ancient times, and today, it is an irreplaceable food ingredient on some Thai menus as well as an active ingredient in modern, scientifically tested medicine (Tamiflu, an anti-influenza drug).
To conclude, the findings in this study indicate that 10% EO of I. verum provided the highest ovicidal activity against P. americana eggs among the five tested EOs, while 10% C. verum provided the highest repellent activity against P. americana adults. Moreover, they suggest that a formulation of their combination (5% I. verum EO+ 5% C. verum EO) in soybean oil can replace cypermethrin as an equally potent but much safer alternative agent for controlling P. americana populations.

Materials and methods
Schematic diagram summarizing the experimental design. Schematic overview of the experimental design of this current study is shown in Fig. 1.  Table 1 25,27 . In brief, 1,000 g of each plant part was cleaned, cut into small pieces, crushed, and extracted of its EO. Water to plant material ratio in the hydro-distillation process was 2:1 ratio, the rate of distillation was about two drops of EO per second. The extraction was completed in 5-6 h. Each EO was collected in 50 ml brown airtight vials and preserved at 4 °C for further bioassay and chemical constituent analysis. Each EO and a combined formulation of the EO with the highest ovicidal activity as well as the EO with the highest repellent activity were prepared into 10% solutions in soybean oil and in ethyl alcohol. The chosen concentration of these EOs and the combined formulation were already proven effective against adult P. americana and adult M. domestica in previous studies by Sittichok et al. 20 , Aungtikun et al. 25 , and Sittichok and Soonwera 47 . All formulations were stored under laboratory conditions (27 ± 2 °C and 70 ± 3% RH) until they were assayed (Fig. 3).   where U is the total number of hatched eggs that were not treated with an active substance and T is the total number of hatched eggs treated with an EO solution or cypermethrin.

Chemical constituent analysis.
(1) Inhibition rate (%) = [U − T/U] × 100,   27 as an index of efficacy comparison between an EO and cypermethrin, was calculated by the formula below, IRI <1 signifies that the EO formulation was less toxic than cypermethrin; IRI = 1 signifies that the EO formulation was equally toxic to cypermethrin; and IRI >1 signifies that the EO formulation was more toxic than cypermethrin.
(2) Inhibition Rate Index (IRI) = % Inhibition rate of each EO formulation % Inhibition rate of cypermethrin www.nature.com/scientificreports/ Repellent activity bioassay. The repellent activity of each EO formulation against P. americana adults was determined by a dual-choice application assay 47 . In the assay, each treatment formulation was dropped onto, absorbed by, and contained in half pieces of Whatman No.1 ® filter paper (18.0 cm wide × 28.0 cm long). One piece was a non-treatment area for comparison with another piece of the treatment area. Two milliliters of clean water were dropped onto a non-treatment piece, and two milliliters of each treatment formulation were dropped onto a treatment piece. Both pieces were placed in a black insectary box (20.0 cm wide × 30.0 cm long × 12.0 cm high) in separate areas: treatment and non-treatment areas. Two plastic cups (5.0 cm in diameter × 4.0 cm high) containing food (2 g of dog biscuits) and drink (50 mL of 10% glucose solution) for the cockroaches were placed on top of each treatment and non-treatment filter paper piece. Five male adults and five female adults of P. americana were released at the center of the insectary box. Either in soybean oil or ethyl alcohol solvent, 10% w/v cypermethrin was used as a positive control for the corresponding treatments of every EO in soybean oil and in ethyl alcohol. Each experiment was repeated ten times. The number of cockroaches situated in the non-treatment area was observed and recorded at 1, 6, 12, 24, and 48 h. The repellent rate was calculated by the following formula 18 : where T is the number of P. americana situated in the treatment area at the time of observation, and N is the number of P. americana situated in the non-treatment area.
The effective repellent index was calculated by the following formula 18 : ERI <1 signifies that the EO formulation was less repellent against P. americana than cypermethrin; ERI = 1 signifies that the EO formulation was equally repellent to cypermethrin; and ERI >1 signifies that the EO formulation was more repellent than cypermethrin.
Statistical analysis. The numbers of hatched eggs and unhatched eggs in the treatment and non-treatment areas were analyzed for significant differences at P<0.05 by a paired t-test method. The repellent activity and ovicidal assays were completely randomized design. Significance differences at P<0.05 were determined by ANOVA (one-way analysis of variance) and Tukey's post hoc test. The coefficients of the regression equation of time versus % repellent activity were determined by SPSS statistical software.

Data availability
All data generated or analyzed during this study are included in this published article [and its supplementary information files].